This proposed research addresses the catalytic mechanism and physiological role of the prolyl endopeptidase (PEPase) of the hyperthermophilic Pyrocossus furiosus. Prolyl endopeptidases are found in such diverse phylogenetic groups as bacteria, plants, fungi, and mammals, yet their function is unknown. It has been hypothesized that prolyl endopeptidases have a role in regulating the half-life of mammalian neuropeptides such as oxytocin, vasopressin, and angiotensin II, since they cleave and inactivate these peptides. The identification of a prolyl endopeptidases in P. furiosus, an archaeon with an optimal growth temperature of 100 deg C, demonstrates that these enzymes function in organisms that inhabit vastly different habitats. The prpA gene from P. furiosus has been cloned into pET 11d and overexpressed in E. coli. The availability of the recombinant protein greatly facilitates characterization of PEPase, as the enzyme is easily purified in high yield from E. coli, and this system avoids the difficulties associated with the large culture volumes of P. furiosus necessary to purify significant quantities of PEPase. In addition, the enzyme is highly thermostable and resists degradation at room temperature. The stated goals of the research include characterizing substrate specificity by measuring Kcat/km values with a series of substrates, studying the catalytic mechanisms of PEPase, particularly with respect to the existence of charge-relay systems characteristic of serine proteases such as alpha-chymotrypsin, assessing the ability to cleave bioactive mammalian peptides, identifying endogenous substrate from P. furiosus cell extracts, and involving undergraduates in a challenging research experience.